Inflammatory monocytes are essential for Innate immune defense against bacterial, fungal, protozoal and viral pathogens. To be effective, monocytes must be recruited from the bone marrow to sites of infection. The mechanisms tiiat drive monocyte recruitment, however, are poorly understood and are the focus of our proposed studies. Our laboratory has demonstrated that the CCR2 chemokine receptor and its ligands MCP1 and MCP3 are required for monocyte emigration from the bone marrow during Listeria monocytogenes infection. How infection in the spleen and liver induces monocyte emigration from the bone marrow, however, remains mysterious. The goal ofthe proposed studies is to determine how infections and circulating microbial molecules promote monocyte emigration from the bone marrow into the bloodstream. Our first aim is to characterize expression of MCPl by cells in the bone marrow following systemic LPS injection and during Listeria monocytogenes infection. These studies will use a recentiy generated MCPl reporter mouse strain and a mouse strain in which MCP1 can be conditionally deleted. Our proposed experiments will determine whether specific deletion of MCPl in these cell populations impairs monocyte trafficking. The second specific aim is to investigate in vivo MCP3 production in bone marrow and peripheral tissues and determine its role in monocyte recruitment. We will generate a conditional MCP3 knockout/reporter mouse strain to determine which cells in bone marrow and peripheraltissuesorchestrate monocyte migration by expressing MCP3. The third specific aim is to investigate the roles of TLR ligands and inflammatory cytokines as inducers of MCP1 and MCP3 expression by stromal and hematopoietic cells in the bone marrow. We will investigate the induction of MCPl and MCP3 in response to LPS or L. monocytogenes infection in reporter mice lacking MyD88, TNF, type I interferon receptor and IFN-gamma. Our studies will provide fundamental insights into the in vivo mechanisms that promote monocyte recruitment to sites of inflammation.